1. Field of the Invention
This invention generally relates to the field of cancer treatment, therapeutics and diagnostics. More specifically, the invention describes antibodies and a method useful for increasing the radiation sensitivity of cancer cells. The invention also provides methods of designing inhibitors of DNA-PKcs that are more specific and result in less harmful side effects.
2. Description of the Related Art
In the clinical setting, the two most common treatments for cancer patients are a drug regimen or treatment with high doses of radiation, or a combination of both. Both approaches kill cancerous (and healthy) cells through a common mechanism of inducing DNA damage. DNA double-strand breaks (DSB) are the most common type of DNA damage resulting from either treatment. In human cells, DNA DSBs are repaired mainly by, the non-homologous end-joining pathway (NHEJ). The DNA-dependent protein kinase complex (DNA-PK) is a key player in the repair of DNA DSBs by this pathway, if DNA-PK is defective, cells are unable to repair DNA DSBs, and thus become highly sensitive to the effects of ionizing radiation and of various cancer drugs. Since DNA-PK is a protein kinase, it is able to transfer phosphate groups to target proteins, and thereby regulate their function. DNA-PK is a protein complex consisting of its DNA-binding and regulatory subunit, which is the Ku protein, and the catalytic subunit, called DNA-PKcs. In the presence of DNA DSBs, Ku binds to the ends of the DNA and recruits DNA-PKcs to the site of the DSB. Once bound to Ku and DNA, DNA-PKcs becomes activated and is capable of phosphorylating target proteins.
Although the biochemical properties of DNA-PK have been extensively studies in vitro, very little is known about how DNA-PK functions in vivo in relation to the repair of DNA DSBs. This lack of progress in studying the physiological functions of DNA-PK is in part due to the unavailability of the right tools or assays to evaluate DNA-PK in vivo activity.
Currently, one of the most commonly used methods to study DNA repair proteins is by immunofluorescence with an antibody to the protein of interest. In response to DNA damage, many of the DNA repair proteins form “foci” that can be visualized with antibodies. It is generally believed that these DNA damage-induced foci correspond to sites where the damages DNA is actively being repaired.
It is currently not possible to detect DNA-PK foci with the antibodies available because DNA-PK is quite abundant in the nucleus, thus when one performs immunofluorescence with any of the available antibodies, the entire nucleus will produce a signal, making it impossible to see any discernable foci. Therefore, it is of interest to develop an antibody that can overcome the problem associated with a very high back-ground signal and can recognize the phosphorylated form of DNA-PKcs when bound to site of DNA DSBs.
DNA-PK is a serine/threonine protein kinase that in vitro is activated by DNA ends and has long been established to play an important role in the repair of DNA double-strand breaks (DSB) by the NHEJ pathway (Smith and Jackson, Genes Dev. 1999 Apr. 15;13(8):916-34). DNA-PK is capable of autophosphorylating the two Ku subunits, Ku70 and Ku80 according to Chan et al., Biochemistry 1999 Feb. 9;38(6):1819-28. Autophosphorylation of DNA-PKcs causes it to dissociate from Ku, resulting in the loss of kinase activity (Chan and Lees-Miller, J Biol Chem. 1996 Apr. 12;271(15):8936-41). in addition, the inventors have shown that the kinase activity of DNA-PKcs is absolutely required for its function in the NHEJ pathway since a DNA-PKcs-deficient CHO cell line expressing a kinase dead form of DNA-PKcs was incapable of repair (Kurimasa et al., The Journal of Immunology, 2000, 165: 3883-3889). Therefore, the kinase activity of DNA-PK is absolutely required for the repair of DNA DSBs; however, the molecular mechanism of this requirement for kinase activity remains to be elucidated. DNA-PK is also capable of autophosphorylation, that is, it transfers phosphate groups onto itself, and that autophosphorylation may be an important mechanism for regulating its kinase activity (Kurimasa et al., Molecular and Cellular Biology, May 1999, p. 3877-3884, Vol. 19, No. 5).
DNA-PKcs is an extremely large protein consisting of 4129 amino acids, and therefore identifying the site of autophosphorylation is comparable to finding a very small needle in a large haystack. Cloning of the DNA-PKcs cDNA is difficult, since the cDNA exceeds 13 kb. In the past, using classical biochemical techniques, several labs have attempted but failed to identify the autophosphorylation sites. For example, in vivo radiolabelling with 32P and 2-dimensional phosphopeptide mapping failed to identify any autophosphorylation sites.
One goal of radiation biology is to find ways to increase the radiation sensitivity of cancer cells. If this could be achieved, it would then be possible to treat cancer patients with lower doses of radiation and thereby dramatically decrease the side effects and complications associated with radiation treatment.
If the site of phosphorylation in DNA-PK could be specifically blocked in cancer cells, for example with a DNA-PKcs inhibitor, then this should inhibit DNA-PKcs-mediated repair of DNA DSBs and thereby increase the radiation sensitivity of the treated cancer cells. Another possible means of increasing radiation sensitivity is the development of therapeutic antibodies that can specifically recognize and bind to the phosphorylated protein.